Lyme disease is a multisystem, tick-borne disease produced by B. burgdorferi. "Lyme Disease," United States, 1991-1992, MMWR 42:345-348 (1993). It is characterized by recurrent systemic and local manifestations involving the skin, heart, nervous system and joints. Lyme disease has three clinical stages. Steere, A. C., "Lyme Disease," N. Engl. J. Med., 321:586-596 (1989). Stage I lasts approximately four weeks, and includes skin rashes such as erythema chronicum and general influenza-like systemic symptoms. An asymptomatic period is followed by Stage II, characterized by musculoskeletal, nervous and cardiac system involvement. Arthritis is the major manifestation of Stage III. The clinical manifestations of Lyme disease are probably due to a mixture of infection by tissue-invasive B. burgdorferi, specific host immune responses to B. burgdorferi antigens, and autoimmune reactions triggered by B. burgdorferi antigens. Steere, A. C., "Lyme Disease," N. Engl. J. Med., 321:586-596 (1989); Duffy, J. "Lyme Disease," Infect. Dis. Clin. N. Am., 1:511-527 (1987); Marcus, L. C., et al., "Fatal Pancarditis In A Patient With Coexistent Lyme Disease and Babesiosis. Demonstration Of Spirochetes In The Myocardium," Ann. Inter. Med., 103:374-376 (1986).
The role of infection by B. burgdorferi in the pathogenesis of Lyme disease is suggested by the presence of B. burgdorferi in tissues (Marcus, L. C., et al., "Fatal Pancarditis In A Patient With Coexistent Lyme Disease and Babesiosis. Demonstration Of Spirochetes In The Myocardium," Ann. Inter. Med., 103:374-376 (1986); Syndman, D. R., et al., "Borrelia Burgdorferi In Joint Fluid In Chronic Lyme Arthritis," Ann. Inter. Med., 104:798-800 (1986)), the strong anti-B. burgdorferi cellular and humoral response in Lyme disease patients (Szczepanski, A., et al., "Lyme Borreliosis: Host Responses to Borrellia burgdorferi," Microbiol. Rev., 55:21-34-22 (1991); Rahn, D. W., "Lyme Disease: Clinical Manifestations, Diagnosis, and Treatment," Sem. Arthritis Rheum., 20:201-218 (1991)), the effectiveness of antibiotic treatment in forestalling clinical manifestations of Stages I, II and III (Steere, A. C., et al., "Successful Parenteral Penicillin Therapy of Established Lyme Arthritis," N. Engl. J. Med., 308:733-740 (1985); Steere, A. C., et al., "Treatment Of The Early Manifestations of Lyme Disease," Ann. Inter. Med., 99:22-26 (1983)). Infection with B. burgdorferi is not the entire story in Lyme disease pathogenesis. However, processes associated with hypersensitivity and autoimmunity also appear to play an important role in at least some of the chronic manifestations of Lyme disease. Evidence for this includes treatment failure of antibiotics in half of patients with arthritis (Steere, A. C., et al., "Successful Parenteral Penicillin Therapy of Established Lyme Arthritis," N. Engl. J. Med., 308:733-740 (1985); Steere, A. C., et al., "Treatment Of The Early Manifestations of Lyme Disease," Ann. Inter. Med., 99:22-26 (1983)), the presence of anti-B. burgdorferi immune complexes in synovial fluid in the absence of demonstrable organisms (Steere, A. C., et al., "Chronic Lyme Arthritis: Clinical and Immunogenetic Differentiation Form Rheumatoid Arthritis," Ann. Inter. Med., 90:896-901 (1979)), heavy infiltration of the synovia of affected joints with T lymphocytes (Yssel, H., et al., "Borrelia Borgdorferi Activates a T Helper Type 1-Like T Cell Subset In Lyme Arthritis," J. Exp. Med., 174:593-601 (1991); Steere, A. C., et al., "Elevated Levels Of Collagenase And Prostaglandin E2 From Synovium Associated With Erosion Of Cartilage And Bone In A Patient With Chronic Lyme Arthritis," Arthritis Rheum, 23:591-599 (1983)), histological similarities of Lyme disease arthritis and rheumatoid arthritis (Yssel, H., et al., "Borrelia Borgdorferi Activates a T Helper Type 1-Like T Cell Subset In Lyme Arthritis," J. Exp. Med., 174:593-601 (1991); Steere, A. C., et al., "Elevated Levels Of Collagenase And Prostaglandin E2 From Synovium Associated With Erosion Of Cartilage And Bone In A Patient With Chronic Lyme Arthritis," Arthritis Rheum, 23:591-599 (1983)), and cross-reactivity of B. burgdorferi antigens with human tissue (Fikrig, E., et al., "Serologic Response To The Borrelia Burgdorferi Flagellin Demonstrates An Epitope Common To A Neuroblastoma Cell Line," Proc. Natl. Acad. Sci. USA, 90:183-187 (1993)). B. burgdorferi can also directly stimulate production of cytokines such as interleukins-1, 2, and 6, interferons, and tumor necrosis factor from normal, uninfected lymphoid cells, synovial cells and rat glioma cells. Miller, L. C., et al., "Balance Of Synovial Fluid IL-1.beta. and IL-1 Receptor Antagonist And Recovery From Lyme Arthritis," Lancet, 341:146-148 (1993); Habicht, G. S., et al., "Cytokines And The Pathogenesis Of Neuroborreliosis: Borrelia Burgdorferi Induces Glioma Cells To Secrete Interleukin-6," J. Infect. Dis., 164:568-574 (1991). This could be important for Lyme disease pathogenesis since interleukin-1 can itself stimulate the production of collagenase and prostaglandin E2 by synovial cells and fibroblasts. Steere, A. C., et al., "Elevated Levels Of Collagenase And Prostaglandin E2 From Synovium Associated with Erosion Of Cartilage And Bone In A Patient With Chronic Lyme Arthritis," Arthritis Rheum, 23:591-599 (1983). The occurrence of hypersensitivity and autoimmunity in Lyme disease underscores the need to exclude B. burgdorferi antigens able to trigger these phenomena from any proposed Lyme disease vaccine.
B. burgdorferi are taxonomically and antigenically complex. B. burgdorferi sensu lato consists of at least three genospecies (I, II, III), defined on the basis of genomic and antigenic similarities, and includes other non-classifiable strains (see Table I, below). Branton, G., et al., "Delineation of Borrelia Burgdorferi Sensu Stricto, Borrelia Garinii Sp. Nov., And Group VS461 Associated With Lyme Borreliosis," Int. J. Syst. Bacteriol, 42:378-383 (1992); Canica, M. M., et al., "Monoclonal Antibodies For Identification Of Borrelia Afzelii Sp. Nov. Associated With Late Cutaneous Manifestations Of Lyme Borreliosis," Scand. J. Infect. Dis., 25:441-448 (1993); Belfaiza, J., et al., "Genomic Fingerprinting of Borrelia Burgdorferi Sensu Lato By Pulse-Field Gel Electrophoresis," J. Clin. Microbiol., 31:2873-2877 (1993). B. burgdorferi sensu strictu consists only of genospecies I. Branton, G., et al., "Delineation of Borrelia Burgdorferi Sensu Stricto, Borrelia Garinii Sp. Nov., And Group VS461 Associated With Lyme Borreliosis," Int. J. Syst. Bacteriol, 42:378-383 (1992); Canica, M. M., et al., "Monoclonal Antibodies For Identification Of Borrelia Afzelii Sp. Nov. Associated With Late Cutaneous Manifestations Of Lyme Borreliosis," Scand. J. Infect. Dis., 25:441-448 (1993); Belfaiza, J., et al., "Genomic Fingerprinting of Borrelia Burgdorferi Sensu Lato By Pulse-Field Gel Electrophoresis," J. Clin. Microbiol., 31:2873-2877 (1993). In addition to antigenic variability between strains ((Szczepanski, A., et al., "Lyme Borreliosis: Host Responses To Borrelia Burgdorferi," Microbiol. Rev., 55:21-34-22 (1991); Barbour, A. G., "Biological And Social Determinants Of The Lyme Disease Problem," Infect. Agents Dis., 1:50-61 (1992)), a given strain may show antigenic variations in different hosts, after exposure to host defenses in a single host, and/or after repeated laboratory passages (Barbour, A. G., et al., "Biology Of Borrelia Species," Microbiol. Rev., 50:381-400 (1986)). The antigenic complexity of B. burgdorferi has in turn complicated development of vaccines and diagnostic assays for Lyme disease.
The surface of B. burgdorferi, the bacterial structure that interacts with the mammalian immune system, is composed of a layer of carbohydrates covering an outer sheath or cell membrane. Barbour, A. G., et al., "Biology Of Borrelia Species," Microbiol. Rev., 50:381-400 (1986). This membrane is composed of a variety of proteins including OspA-E, a lipopolysaccharide, and a peptidoglycan. Barbour, A. G., et al., "Biology of Borrelia species," Microbiol. Rev., 50:381-400 (1986); Bergstrom, S., et al., "Molecular Analysis Of Linear Plasmid-encoded Major Surface Proteins, OspA and OspB, Of The Lyme Disease Spirochete Borrelia burgdorferi," Molec. Microbiol., 3:479-486 (1989); Fuchs, R., et al., "Molecular Analysis And Expression Of A Borrelia burgdorferi Gene Encoding A 22kDa Protein (pC) in Escherichia coli," Molec. Microbiol., 6:503-509 (1992); Lam, T. T., et al., "Outer Surface Proteins E and F of Borrelia burgdorferi, The Agent Of Lyme Disease," Infect. Immun., 62:290-298 (1994); Beck, G., et al., "Chemical And Biologic Characterization Of A Lipopolysaccharide From The Lyme Disease Spirochete (Borrelia burgdorferi)," J. Infect. Dis., 152:108-117 (1985); Takayama, K., et al., "Absence of Lipopolysaccharide In The Lyme Disease Spirochete, Borrelia burgdorferi," Infect. Immun., 55:2311-2313 (1987). The existence of the latter two structures in B. burgdorferi is still controversial. Beck, G., et al., "Chemical And Biologic Characterization Of A Lipopolysaccharide From The Lyme Disease Spirochete (Borrelia burgdorferi)," J. Infect. Dis., 152:108-117 (1985); Takayama, K., et al., "Absence of Lipopolysaccharide In The Lyme Disease Spirochete, Borrelia burgdorferi," Infect. Immun., 55:2311-2313 (1987). OspA and OspB lipoproteins are analogous to the VMP proteins of B. hermsii, and appear to undergo extensive variation among B. burgdorferi genotypes and isolates in vitro and in vivo. This variability can be detected by both gene and gene product analysis. Marconi, R. T., et al., "Variability Of osp Genes And Gene Products Among Species of Lyme Disease Spirochetes," Infect. Immun., 61:2611-2617 (1993); Schoberg, R. J., et al., "Identification Of A Highly Cross-reactive Outer Surface Protein B Epitope Among Diverse Geographic Isolates of Borrelia Spp. Causing Lyme Disease," Infect Immun., 32:489-500 (1994). It appears to be responsible for the ability of B. burgdorferi to become resistant to the bactericidal effects of anti-OspA/OspB antibodies. Sadziene, A., et al., "Antibody-resistant Mutants of Borrelia burgdorferi: In Vitro Selection And Characterization," J. Exp. Med., 176:799-809 (1992); Coleman, J. L., et al, "Selection Of An Escape Variant Of Borrelia burgdorferi By Use Of Bactericidal Monoclonal Antibodies To OspB," Infect. Immun., 60:3098-3104 (1992). Antibodies against OspA prevent development of carditis in the scid mouse. Schaible, U. E., et al., "Monoclonal Antibodies Specific For The Outer Membrane Protein A (OspA) Of Borrelia burgdorferi Prevent Lyme Disease Borreliosis In Severe Combined Immunodeficiency (Scid) Mice," Proc. Natl. Acad. Sci. USA, 87:3768-3772 (1990). The immunogenicity of OspA appears to be influenced by its lipidic content (Erdile,, L. F., et al., "Role Of Attached Lipid In Immunogenicity Of Borrelia burgdorferi OspA," Infect. Immun., 61:81-90 (1993)), and, as mentioned above, the protein has been used successfully as a vaccine in animals (Fikrig, E., et al., "Protection Of Mice Against The Lyme Disease Agent By Immunizing With Recombinant OspA," Science, 250:553-556 (1990)). The heterogeneity of these B. burgdorferi plasmid-encoded proteins is not unexpected. We have reported that E. coli plasmid genes appear to have an increased potential for variation as they usually have a higher replication rate than the bacterial chromosome and carry insertion sequences and transposons. Timmis, K., et al., "Instability Of Plasmid Sequences: Macro And Microevolution Of The Antibiotic Resistance Plasmids R6-5," Mol. Gen. Genet., 167:11-19 (1978). All these properties increase the opportunities for emergence of mutations. Timmis, K., et al., "Instability Of Plasmid Sequences: Macro And Microevolution Of The Antibiotic Resistance Plasmids R6-5," Mol. Gen. Genet., 167:11-19 (1978).
B. burgdorferi antigens cross-react with antigens of other pathogenic and human commensal spirochetes and with antigens of Gram-negative bacteria. Rasiah. C., et al., "Purification And Characterization Of A Tryptic Peptide Of Borrelia burgdorferi Flagellin, Which Reduces Cross-reactivity In Immunobolots And ELISA," J. Gen. Microbiol., 138:147-154 (1992); Coleman, J. L., et al., "Characterization Of Antigenic Determinants Of Borrelia burgdorferi Shared By Other Bacteria," J. Infect. Dis., 165:658-656 (1992). B. burgdorferi flagellin has 60-95% amino acid sequence similarity to flagellins from related bacteria (Rasiah. C., et al., "Purification And Characterization Of A Tryptic Peptide Of Borrelia burgdorferi Flagellin, Which Reduces Cross-reactivity In Immunobolots And ELISA," J. Gen. Microbiol., 138:147-154 (1992); Coleman, J. L., et al., "Characterization Of Antigenic Determinants Of Borrelia burgdorferi Shared By Other Bacteria," J. Infect. Dis., 165:658-656 (1992)), 50% to unrelated ones (Rasiah. C., et al., "Purification And Characterization Of A Tryptic Peptide of Borrelia burgdorferi Flagellin, Which Reduces Cross-reactivity In Immunobolots And ELISA," J. Gen. Microbiol., 138:147-154 (1992); Coleman, J. L., et al., "Characterization Of Antigenic Determinants Of Borrelia burgdorferi Shared By Other Bacteria," J. Infect. Dis., 165:658-656 (1992)), and cross-reacts with human tissue antigens as well (Rasiah. C., et al., "Purification And Characterization Of A Tryptic Peptide Of Borrelia burgdorferi Flagellin, Which Reduces Cross-reactivity In Immunobolots And ELISA," J. Gen. Microbiol., 138:147-154 (1992); Magnarelli, L. A., et al., "Comparison Of Whole-cell Antibodies And An Antigenic Flagellar Epitope Of Borrelia burgdorferi In Serologic Tests For Diagnosis Of Lyme Borreliosis," J. Clin. Microbiol., 30:3158-3162 (1992)). In general, there is a broad cross-reactivity of B. burgdorferi proteins with proteins of other bacterial species including Borrelia, Treponema, and gram-negative rods that are capable of inducing anamnestic responses. Coleman, J. L., et al., "Characterization Of Antigenic Determinants Of Borrelia burgdorferi Shared By Other Bacteria," J. Infect. Dis., 165:658-656 (1992). Moreover, the surface of B. burgdorferi may be similar to the surface of T. pallidum, with major membrane proteins anchored by lipids to the outermost cytoplasmic membrane, thus potentially decreasing their immunogenicity. Cox, D. L., et al., "The Outer Membrane, Not A Coat Of Host Proteins, Limits Antigenicity Of Virulent Treponema Pallidum," Infect. Immun., 60:1076-1083 (1992). Current studies utilizing immune responses to identify protective B. burgdorferi antigens as a basis for developing vaccines for Lyme disease have just begun, and their results have been problematic.
An approach to these problems is to examine immunogenic components of B. burgdorferi at the molecular level. Barbour, A. G., "Biological And Social Determinants Of The Lyme Disease Problem," Infect. Agents Dis., 1:50-61 (1992); Bergstrom, S, et al., "Molecular Analysis Of Linear Plasmid-encoded Major Surface Proteins, OspA and OspB, Of The Lyme Disease Spirochete Borrelia burgdorferi," Molec. Microbiol., 3:479-486 (1989). The genetic material of B. burgdorferi consists of double-stranded linear chromosomal DNA with a putative size of 990 kb, and linear and covalently closed DNA plasmids whose sizes range from 7.6 to 70.0 kb. Bergstrom, S., et al., "Molecular Analysis Of Linear Plasmid-encoded Major Surface Proteins, OspA and OspB, Of The Lyme Disease Spirochete Borrelia burgdorferi," Molec. Microbiol., 3:479-486 (1989); Casjens, S., et al., "Linear Chromosomal Physical And Genetic Map Of Borrelia burgdorferi, The Lyme Disease Agent," Molec. Microbiol., 8:967-980 (1993). A 49.0 kb linear plasmid encodes the expression of the immunogenic, polymorphic 31 kDa OspA and 34 kDa OspB outer membrane lipoproteins. These proteins share 53% identity in their amino acid sequences. Bergstrom, S., et al., "Molecular Analysis Of Linear Plasmid-encoded Major Surface Proteins, OspA and OspB, Of The Lyme Disease Spirochete Borrelia burgdorferi," Molec. Microbiol., 3:479-486 (1989); Coleman, J. L., et al, "Selection Of An Escape Variant Of Borrelia burgdorferi By Use Of Bactericidal Monoclonal Antibodies To OspB," Infect. Immun., 60:3098-3104 (1992). Their function in bacterial physiology and role in virulence is still unclear. Bergstrom, S., et al., "Molecular Analysis Of Linear Plasmid-encoded Major Surface Proteins, OspA and OspB, Of The Lyme Disease Spirochete Borrelia burgdorferi," Molec. Microbiol., 3:479-486 (1989); Volkman, D. J., et al., "Characterization Of An Immunoreactive 93-kDa Core Protein Of Borrelia burgdorferi With A Human IgG Monoclonal Antibody," J. Immunol., 146:3177-3182 (1991). Molecular genetic techniques have permitted cloning, expression, and sequencing of the plasmid-encoded OspA-F genes and the genes of other B. burgdorferi protein antigens (i.e. flagellin, p39, IpLA7 and heat-shock proteins). Bergstrom, S., et al., "Molecular Analysis Of Linear Plasmid-encoded Major Surface Proteins, OspA and OspB, Of The Lyme Disease Spirochete Borrelia burgdorferi," Molec. Microbiol., 3:479-486 (1989); Fuchs, R., et al., "Molecular Analysis And Expression Of A Borrelia burgdorferi Gene Encoding A 22 kDa Protein (pC) in Escherichia coli," Molec. Microbiol., 6:503-509 (1992); Lam, T. T., et al., "Outer Surface Proteins E and F of Borrelia burgdorferi, The Agent Of Lyme Disease," Infect. Immun., 62:290-298 (1994). This has opened the way for chemical and structural characterization of their gene products and their purification in large amounts for use as vaccines and diagnostic reagents. Bergstrom, S., et al., "Molecular Analysis Of Linear Plasmid-encoded Major Surface Proteins, OspA and OspB, Of The Lyme Disease Spirochete Borrelia burgdorferi," Molec. Microbiol., 3:479-486 (1989); Fuchs, R., et al., "Molecular Analysis And Expression Of A Borrelia burgdorferi Gene Encoding A 22 kDa Protein (pC) in Escherichia coli," Molec. Microbiol., 6:503-509 (1992); Lam, T. T., et al., "Outer Surface Proteins E and F of Borrelia burgdorferi, The Agent Of Lyme Disease," Infect. Immun., 62:290-298 (1994)); Volkman, D. J., et al., "Characterization Of An Immunoreactive 93-kDa Core Protein Of Borrelia burgdorferi With A Human IgG Monoclonal Antibody," J. Immunol., 146:3177-3182 (1991); Wilske, B., et al., "Immunological And Molecular Polymorphisms Of OspC, An Immunodominant Major Outer Surface Protein Of Borrelia burgdorferi," Infect. Immun., 61:2182-2191 (1993); Hansen, K., et al., "Immunochemical Characterization And Isolation Of The Gene For A Borrelia burgdorferi Immunodominant 60-Kilodalton Antigen Common To A Wide Range Of Bacteria," Infect. Immun., 56:2047-2053 (1986). Little is known, however, about the role of OspA-F, flagella, the 60 kDa and 73 kDa proteins, or any B. burgdorferi antigens, for that matter, in infection of ticks or production of disease in mammals during the different stages of Lyme disease. Barbour, A. G., "Biological And Social Determinants Of The Lyme Disease Problem,"Infect. Aqents Dis., 1:50-61 (1992).
The infected host develops immune responses to B. burgdorferi outer surface membrane antigens, periplasmic antigens such as flagella, and cytoplasmic antigens such as heat-shock proteins. Rasiah, C., et al., "Purification And Characterization Of A Tryptic Peptide Of Borrelia burgdorferi Flagellin, Which Reduces Cross-reactivity In Immunobolots And ELISA," J. Gen. Microbiol., 138:147-154 (1992); Hansen, K., et al., "Immunochemical Characterization And Isolation Of The Gene For A Borrelia burgdorferi Immunodominant 60-Kilodalton Antigen Common To A Wide Range Of Bacteria," Infect. Immun., 56:2047-2053 (1986). Such responses have been detected by ELISA, IFA, immunoblotting, and T cell mitogenesis. Krause, A., et al., "Cellular Immune Reactivity To Recombinant OspA And Flagellin From Borrelia Burgdorferi In Patients With Lyme Borreliosus," J. Clin. Invest., 90:1077-1084 (1992); Fikrig, E., et al., "Serologic Diagnosis Of Lyme Disease Using Recombinant Outer Surface Proteins A And B And Flagellin," J. Infect. Dis., 165:1127-1132 (1992). Some of the humoral response comprises protective antibodies that can inhibit B. burgdorferi growth and/or are bactericidal. Pavia, C. S., et al., "Antiborrelial Activity Of Serum From Rats Injected With The Lyme Disease Spirochete," J. Infect. Dis., 163:656-659; Sadziene, A., et al., "In Vitro Inhibition of Borrelia burgdorferi Growth By Antibodies," J. Infect. Dis., 167:165-172 (1993); Fikrig, E., et al., "Long-Term Protection Of Mice From Lyme Disease By Vaccination With OspA," Infect. Immun., 60:773-777 (1992). However, there are wide individual variations in human serological response to B. burgdorferi (Barbour, A. G., "Biological And Social Determinants Of The Lyme Disease Problem," Infect. Agents Dis., 1:50-61 (1992); Corpuz, M., et al., "Problems In The Use Of Serologic Tests For The Diagnosis Of Lyme Disease," Ann. Inter. Med., 151:1837-1840 (1991)), and high titers of specific antibodies may be demonstrable only late in the evolution of disease (Barbour, A. G., "Biological And Social Determinants Of The Lyme Disease Problem," Infect. Agents Dis., 1:50-61 (1992); Corpuz, M., et al., "Problems In The Use Of Serologic Tests For The Diagnosis Of Lyme Disease," Ann. Inter. Med., 151:1837-1840 (1991)). Cross reactivity of B. burgdorferi antigens with host antigens may induce host tolerance and host autoimmunity. Fikrig, E., et al., "Serologic Response To The Borrelia Burgdorferi Flagellin Demonstrates An Epitope Common To A Neuroblastoma Cell Line," Proc. Natl. Acad. Sci. USA, 90:183-187 (1993). The small numbers of bacteria apparently found in tissues and fluids in Lyme disease may conspire against a robust humoral immune response. Duray, P. H., "Capturing Spirochetes From Humans," Am. J. Clin. Pathol., 99:4-6 (1993); Dattwyler, R. J., et al., "Seronegative Lyme Disease. Dissociations of Specific T- and B-lymphocytes Responses to B. burgdorferi," N. Engl. J. Med., 319:141-1446 (1988)), and the possible intracellular location of the bacteria in macrophages or other immunologically hidden sites may exacerbate this situation (Montgomery, R. R., et al., "The Fate Of Borrelia burgdorferi, The Agent For Lyme Disease, In Mouse Macrophages," J. Immunol, 150:909-915 (1993)). Furthermore, some patients show dissociation between humoral and cell-mediated immune responses, with a lack of serological response to infection in the presence of strong T cell reactivity to B. burgdorferi antigens (Lahesmas, R., et al., "Preferential Use Of T Cell Antigen Receptor V Region Gene Segment Vbeta5.1 By Borrelia Burgdorferi Antigen-reactive T Cell Clones Isolated From A Patient With Lyme Disease," J. Immunol., 150:4125-4135 (1993)).
Host immune response to B. burgdorferi is influenced by genetic factors in the host as well as by the antigens expressed by the infecting B. burgdorferi strain. In human beings, immune response to B. burgdorferi and development of clinical manifestations of Lyme disease can depend on HLA haplotypes. Golde, W. T., et al., "The Major Histocompatibility Complex-restricted Response Of Recombinant Inbred Strains Of Mice To Natural Tick Transmission Of Borrelia burgdorferi," J. Exp. Med., 177:9-17 (1993). In mice, disease-susceptible (C3H) and disease-resistant (BALB/c) inbred strains have been found (De Souza, M. S., et al., "Long-Term Study Of Cell-Mediated Responses to Borrelia burgdoferi In The Laboratory Mouse," Infect. Immun., 61:1884-1822 (1993)), and patterns of antibody response to B. burgdorferi antigens are MHC-restricted in mice infected by natural tick transmission of B. burgdorferi (Golde, W. T., et al., "The Major Histocompatibility Complex-restricted Response Of Recombinant Inbred Strains Of Mice To Natural Tick Transmission of Borrelia burgdorferi," J. Exp. Med., 177:9:17 (1993)).
Study and interpretation of the humoral and cellular immune responses to B. burgdorferi antigens in Lyme disease is complicated by the complex mixes of B. burgdorferi antigens used in these studies and the absence of antigenic standardization. Barbour, A. G., "Biological And Social Determinants Of The Lyme Disease Problem," Infect. Agents Dis., 1:50-61 (1992). ELISA results may vary because of fluctuations in amounts of particular B. burgdorferi antigens bound to solid phases from the diverse antigenic mixtures of whole cell sonicates. Schwartz, B. S., et al., "Antibody Testing In Lyme Disease: A Comparison Of Results In Four Laboratories," JAMA, 262:3431-3434 (1989); Luger, S. W., et al., "Serologic Tests For Lyme Disease: Interlaboratory Variability," Arch. Intern. Med., 150:761-763 (1990); Bakken. L. L., et al., "Performance of 45 Laboratories Participating In A Proficiency Testing Program For Lyme Disease Serology," JAMA, 268:891-895 (1992). In immunoblots, the apparent lack of reactivity of a serum with given B. burgdorferi antigens may be due to an inability to resolve antigens of similar molecular weight (Karlsson, M, et al., "Comparison Of Western Blot And Enzyme-linked Immunosorbent Assay For Diagnosis Of Lyme Borreliosis," Eur. J. Clin. Microbiol. Infect. Dis., 8:871-877 (1989); Aron-Hott, L., et al., "Lipopolysaccharide-independent Radioimmunoprecipitation And Identification Of Structural And in vivo Induced Immunogenic Surface Proteins of Salmonella typhi In Typhoid," Vaccine, 11:10-17 (1993)), to denaturation of conformational epitopes produced by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Karlsson, M., et al., "Comparison Of Western Blot And Enzyme-linked Immunosorbent Assay For Diagnosis Of Lyme Borreliosis," Eur. J. Clin. Microbiol. Infect. Dis., 8:871-877 (1989); Aron-Hott, L., et al., "Lipopolysaccharide-independent Radioimmunoprecipitation And Identification Of Structural And in vivo Induced Immunogenic Surface Proteins of Salmonella typhi In Typhoid," Vaccine, 11:10-17 (1993)), or to low antigenic concentration in the sample (Karlsson, M., et al., "Comparison Of Western Blot And Enzyme-linked Immunosorbent Assay For Diagnosis Of Lyme Borreliosis," Eur. J. Clin. Microbiol. Infect. Dis., 8:871-877 (1989); Aron-Hott, L., et al., "Lipopolysaccharide-independent Radioimmunoprecipitation And Identification Of Structural And in vivo Induced Immunogenic Surface Proteins of Salmonella typhi In Typhoid," Vaccine, 11:10-17 (1993)). Cross-contamination of one B. burgdorferi antigen with another in SDS-PAGE and non-specific binding of immunoglobulins to denatured B. burgdorferi proteins can also interfere with the interpretation of immunoblots. Karlsson, M, et al., "Comparison Of Western Blot And Enzyme-linked Immunosorbent Assay For Diagnosis Of Lyme Borreliosis," Eur. J. Clin. Microbiol. Infect. Dis., 8:871-877 (1989); Aron-Hott, L., et al., "Lipopolysaccharide-independent Radioimmunoprecipitation And Identification Of Structural And in vivo Induced Immunogenic Surface Proteins of Salmonella typhi In Typhoid," Vaccine, 11:10-17 (1993).
Protection against B. burgdorferi infection and subsequent development of Lyme disease can be achieved by immunization with killed B. burgdorferi cells or with purified antigens of this bacteria. Fikrig, E., et al., "Protection Of Mice Against The Lyme Disease Agent By Immunizing With Recombinant OspA," Science, 250:553-556 (1990); Fikrig, E., et al., "Long-Term Protection Of Mice From Lyme Disease By Vaccination With OspA," Infect. Immun., 60:773-777 (1992); Fikrig, et al., "OspA Vaccination Of Mice With Established Borrelia burgdoferi Infection Alters Disease But Not Infection," Infect. Immun., 61:2553-2557 (1993). Adoptive transfer of T cells from chronically-infected C3H mice failed to prevent infection and disease development in recipient C3H mice. De Souza, M. S., et al., "Long-Term Study Of Cell-Mediated Responses to Borrelia burgdoferi In The Laboratory Mouse," Infect. Immun., 61:1884-1822 (1993). These observations are consistent with induced protection being primarily mediated by antibodies against borrelial antigens, and not by T cell-mediated cellular immune responses. Fikrig, E., et al., "Protection Of Mice Against The Lyme Disease Agent By Immunizing With Recombinant OspA," Science, 250:553-556 (1990); Fikrig, E., et al., "Long-Term Protection Of Mice From Lyme Disease By Vaccination With OspA," Infect. Immun., 60:773-777 (1992). Antisera against B. burgdorferi OspA/OspB proteins have been used to provide passive protection against B. burgdorferi infection in scid and C3H/HeJ mice; immunization of these mouse strains with rOspA/rOspB protein provides active protection. Schaible, U. E., et al., "Monoclonal Antibodies Specific For The Outer Membrane Protein A (OspA) Of Borrelia burgdorferi Prevent Lyme Disease Borreliosis In Severe Combined Immunodeficiency (Scid) Mice," Proc. Natl. Acad. Sci. USA, 87:3768-3772 (1990); Fikrig, E., et al., "Long-Term Protection Of Mice From Lyme Disease By Vaccination With OspA," Infect. Immun., 60:773-777 (1992); Fikrig, et al., "OspA Vaccination Of Mice With Established Borrelia burgdoferi Infection Alters Disease But Not Infection," Infect. Immun., 61:2553-2557 (1993). In scid mice, however, passive protection appears to be complete only for infection by B. burgdorferi expressing identical or very similar OspA. Schaible, U. E., "Immune Sera To Individual Borrelia burgdoferi Isolates Or Recombinant OspA Thereof Protect SCID Mice Against Infection with Homologous Strains But Only Partially Or Not At All Against Those Of Different OspA/OspB Genotype," Vaccine, 11:1049-1054 (1993). This is due to variation in the genes and gene products of the OspA/OspB operon among various B. burgdorferi genotypes and different isolates of the same genotype. Marconi, R. T., et al., "Variability Of osp Genes And Gene Products Among Species of Lyme Disease Spirochetes," Infect. Immun., 61:2611-2617 (1993). Similarly, vaccination of C3H/HeJ mice with truncated rOspA fails to elicit protective immunity, while B. burgdorferi expressing truncated OspB are able to escape immune destruction in mice vaccinated with OspB. Bockenstedt, L. K., et al., "Inability Of Truncated Recombinant OspA Proteins To Elicit Protective Immunity To Borrelia burgdoferi In Mice," J. Immunol., 151:900-906 (1993); Fikrig, E., et al., "Evasion Of Protective Immunity By Borrelia burgdoferi By Truncation Of Outer Surface Protein B.," Proc. Nat. Acad. Sci. USA, 90:4092-4096) (1993). B. burgdorferi variants lacking expression of OspA/OspB are still able to confer protection to immunized mice even against challenges with strains expressing OspA/OspB antigens. Norton Hughes, C. A., et al., "Protective Immunity Is Induced By A Borrelia burgdoferi Mutant That Lacks OspA and OspB," Infect. Immun., 61:5151-5122 (1993). These experiments suggest that plasmid-encoded OspA/OspB are not the only B. burgdorferi antigens that can confer protection. Norton Hughes, C. A., et al., "Protective Immunity Is Induced By A Borrelia burgdoferi Mutant That Lacks OspA and OspB," Immun., 61:5151-5122 (1993). Another property of plasmids that makes plasmid-encoded gene products less suitable for use in vaccines is the non-essential nature of plasmids to bacterial survival, because they are lost during cell division with relatively high frequency. Timmis, K., et al., "Instability Of Plasmid Sequences: Macro And Microevolution Of The Antibiotic Resistance Plasmids R6-5," Mol. Gen. Genet., 167:11-19 (1978). For example, vaccination of animal and human populations with B. burgdorferi plasmid-encoded OspA/OspB could potentially select for the emergence and dissemination of strains expressing different OspA/OspB proteins or lacking the expression of these dispensable proteins altogether. Timmis, K., et al., "Instability Of Plasmid Sequenced: Macro And Microevolution Of The Antibiotic Resistance Plasmids R6-5," Mol. Gen. Genet., 167:11-19 (1978); Schaible, U. E., "Immune Sera To Individual Borrelia burgdoferi Isolates Or Recombinant OspA Thereof Protect SCID Mice Against Infection with Homologus Strains But Only Partially Or Not At All Against Those Of Different OspA/OspB Genotype," Vaccine, 11:1049-1054 (1993); Bockenstedt, L. K., et al., "Inability Of Truncated Recombinant OspA Proteins To Elicit Protective Immunity To Borrelia burgdoferi In Mice," J. Immunol., 151:900-906 (1993); Norton Hughes, C. A., et al., "Protective Immunity Is Induced By A Borrelia burgdoferi Mutant That Lacks OspA and OspB," Infect. Immun., 61:5151-5122 (1993).
In view of the above-noted deficiencies in the art, the need remains for improved procedures to detect and treat Lyme disease. The present invention is directed to meeting this objective.